The .beta.-lactamases are a group of enzymes which are produced by certain bacteria and confer resistance to .beta.-lactam antibiotics. Such resistance has implications in the therapy of bacterial infections with .beta.-lactam antibiotics, since bacterial resistance to the antibiotic may be of sufficient magnitude to cause the therapy to fail. It is therefore desirable to have available accurate and sensitive assays for the presence of .beta.-lactamases in order to select an appropriate antibiotic therapy for a patient and to monitor patients receiving .beta.-lactam therapy for the emergence of bacterial resistance due to production of .beta.-lactamases.
.beta.-lactamases confer resistance by hydrolysis of the .beta.-lactam ring of the antibiotic. Ring hydrolysis results in inactivation of the .beta.-lactam antibiotic. Enzymatic hydrolysis is believed to be initiated by the attack of a serine hydroxyl in the active site of the enzyme which opens the .beta.-lactam ring and produces an acyl-enzyme intermediate. The acyl intermediate may be broken down by either one of two pathways depending on the stability and steric bulk of the leaving group. The two pathways of breakdown of the acyl intermediate are illustrated in FIG. 1. If the leaving group, X-Y, cannot be eliminated or is eliminated only very slowly the breakdown of the acyl-enzyme intermediate proceeds by pathway A in which the leaving group, X-Y, is eliminated after the enzyme is deacylated. This results in a high turnover of enzyme substrate (i.e., the antibiotic) and is therefore desirable for development of a sensitive detection system for .beta.-lactamases.
When the leaving group is eliminated rapidly, the breakdown of the acyl-enzyme intermediate occurs by pathway B in which the elimination occurs before the enzyme is deacylated. This produces a more stable acyl-enzyme intermediate which hydrolyzes slowly to regenerate free enzyme. Incubation of .beta.-lactamase with substrates of this type results in an initial burst of enzyme activity, followed by .beta.-lactamase being nearly completely inactivated.
Elimination of the leaving group may not occur exclusively by only one of these alternative pathways. .beta.-lactamase substrates which have a 3' leaving group generally are believed to be hydrolyzed to varying extents by both pathways.
Research in the field of synthesizing substrates for .beta.-lactamase has primarily concentrated on developing compounds which are hydrolyzed by pathway B (FIG. 1) which results in enzyme inactivation. Most of the known chromogenic substrates for .beta.-lactamase have been synthesized by modification of cephalosporins and have a 3' leaving group which is eliminated during hydrolysis of the lactam ring. These compounds tend to inhibit enzyme activity or deactivate the enzyme and therefore allow .beta.-lactam antibiotics to inhibit the growth of the bacteria. However, such substrates have the disadvantage of reduced sensitivity when used in assays for .beta.-lactamase as compared to substrates such as those herein disclosed.
While not wishing to be bound by any particular reaction mechanism, Applicants believe that the .beta.-lactamase substrates of the present invention are hydrolyzed primarily by the first pathway, since the leaving groups are relatively bulky and would be eliminated slowly. Once cleaved, the leaving groups are fluorescent, visible or capable of producing a visually detectable color in an assay. These compounds therefore provide improved assay sensitivity over conventional reagents and, in addition, can be detected using fluorometric and spectrophotometric detectors commonly available in clinical laboratories. In particular, the inventive fluorogenic substrates represent an improvement over prior art .beta.-lactamase substrates in that they fluoresce at a wavelength range which can be detected using such automated fluorescence detecting systems, making assays using such substrates more practical and economical when a large number of tests are to be performed, for example in a clinical laboratory. The inventive substrates which are capable of producing visually detectable colors upon cleavage provide an advantage over previously known .beta.-lactamase substrates in that the leaving group can be further reacted to produce an insoluble dye which can be precipitated on a solid surface (e.g., a membrane) where it can be easily visualized.
Substrates for .beta.-lactamase which are nonfluorescent prior to cleavage of the .beta.-lactam ring and fluorescent after cleavage are described in U.S. Pat. No. 4,965,193 to Chen and U.S. Pat. No. 4,740,459 to Chen et al. The disclosed substrates are B-lactam antibiotics having an acyl side chain containing an .alpha.-amino group and an .alpha.-phenyl group or its derivatives. In the presence of .beta.-lactamase, the amide bond is cleaved and the resulting open-ring compound fluoresces. In U.S. Pat. No. 4,965,193 it is further disclosed that fluorescence can be improved using a fluorescence developer which consists of a mild oxidizing agent and formaldehyde. These compounds differ from those of the present invention by virtue of the fact that the antibiotic itself becomes fluorescent when the .beta.-lactam ring is opened, whereas the fluorogenic compounds of the invention release a leaving group which fluoresces. The substrates of the invention are therefore useful in fluorescence assays regardless of the structure of the acyl side chain. In addition, the inventive fluorogenic substrates require no post cleavage chemical treatment and are therfore amenable to providing kinetic information.
Chromogenic precipitating substrates for .beta.-lactamase are disclosed in U.S. Pat. No. 4,978,613 to Bieniarz et al. These substrates include a moiety, R.sub.2, linked to a .beta.-lactam compound by a thioester linkage. This moiety, together with the sulfur atom to which it is attached, forms a sulfur-containing leaving group, S-R.sub.2, the conjugate acid of which has a pK.sub.a less than 8. The leaving group is eliminated upon cleavage of the .beta.-lactam ring by .beta.-lactamase and reacts with a tetrazolium salt to form a colored precipitate without further breakdown of S-R.sub.2. The chromogenic substrates of Bieniarz et al. are therefore limited to chromogens which have a sulfur atom for formation of the thioether or thioester linkage to the .beta.-lactam compound. In contrast, the .beta.-lactamase substrates of the present invention contain carbamate, carbonate, thiocarbamate or thiocarbonate linkages and can therefore be synthesized using non-sulfur-containing chromogens.
U.S. Pat. No. 3,355,452 and U.S. Pat. No. 3,484,437 to Urech et al. disclose derivatives of 7-amino-cephalosporanic acid which are synthesized by reaction of desacetyl-7--amino-cephalosporanic acid with an isocyanic ester. These semi-synthetic antibiotics, however, do not include a chromogenic or fluorescent leaving group and are resistant to cleavage by penicillinases and cephalosporinases. They are therefore not suitable as substrates for use in assays for .beta.-lactamase such as those assays presently described.